The exquisite specificity of antibody-antigen interactions is ideal for therapeutic applications, bt the therapeutic use of antibodies has been limited to extracellular targets because of limited access of antibodies into cells. We developed a cell-penetrating antibody, mAb 3E10, as an intracellular transporter to deliver antibodies into cells as bispecific single-chain Fv fragments (scFv). mAb 3E10 is unique in that it penetrates cells without apparent harm and has been administered to humans without evidence of toxicity. We chose Mdm2 as a target because Mdm2 is an important target in cancer therapy, and we selected mAb 3G5 to target the N-terminus of MDM2. Our long-term goal is to develop 3E10-3G5 bispecific scFv as a therapeutic for the treatment of cancer and extend the application of 3E10 to target intracellular sites that are currently considered undruggable with small molecules. The objective here in the pursuit of this goal is to establish proof-of-principle for targeting antibodies to intracellular targets by developing and evaluating 3E10-3G5 as a model system. Our central hypothesis is that 3E10-3G5 scFv will be therapeutically effective in many cancers, and it will synergize with conventional cancer therapies. The rationale for the proposed research is that intracellular antibodies can target intracellular epitopes that are undruggable and cannot be targeted with small molecules, and they have greater binding specificity than small molecules, thus eliminating off-target binding responsible for toxicity of therapy based on small molecule inhibitors. Specific aims: 1) Examine a broad spectrum of human tumor xenografts to determine which cancer cell types are responsive to 3E10-3G5 scFv, 2) Elucidate the p53-dependent and p53-independent mechanisms responsible for inhibiting growth of cancer cells responsive to 3E10-3G5 scFv, 3) Determine the clinical efficacy of 3E10-3G5 in combination with conventional chemotherapy in human tumor xenografts in vivo. The proposed research is significant because our intracellular transport system can be used to transport antibodies into cells to target epitopes on transcription factors and DNA repair proteins that cannot be targeted with small molecules, thus broadening the spectrum of potential intracellular targets for cancer therapy. The proposed research is innovative because it utilizes a novel cell-penetrating antibody that serves as a transport system to deliver antibodies directly into cells through an equilibrative nucleoside salvage pathway. Our laboratory is the only one to develop this technology, and we have demonstrated its feasibility and clinical efficacy in vitro and in vivo.